Cytochrome P450 monooxygenase from the anaerobic microorganism Clostridium acetobutylicum (CYP 152A2) has been produced in Escherichia coli. CYP152A2 was shown to bind a broad range of saturated and unsaturated fatty acids and corresponding methyl esters and demonstrated a high peroxygenase activity of up to 200 min(-1) with myristic acid. Although a high concentration of hydrogen peroxide of 200 pM was necessary for high activities of the enzyme, it led to a fast enzyme inactivation within 2-4 min. This might reflect the natural function of CYP152A2 as a rapid hydrogen peroxide scavenging enzyme. In two different reconstituted systems with NADPH, CYP152A2 was able to convert 10 times more substrate, if provided with flavodoxin and flavodoxin reductase from E coli and even 3040 times more substrate with the CYP102A1 -reductase from Bacillus megaterium. According to the clear preference for hydroxylation at alpha-posftion, CYP152A2 can be referred to as fatty acid cc-hydroxylase. (c) 2007 Elsevier Inc. All rights reserved.